Proximity assays are used to assess whether two particular proteins or portions thereof are in close proximity, e.g., proteins that are bound to each other, fusion proteins, and/or proteins that are positioned in close proximity. One such assay, known as proximity ligation assay (PLA), features two antibodies (raised in different species) bound to the targets of interest (see Nature Methods 3, 995-1000 (2006)). PLA probes, which are species-specific secondary antibodies with a unique oligonucleotide strand attached, are then bound to the appropriate primary antibodies. In the case of the targets being in close proximity, the oligonucleotide strands of the PLA probes can interact with additional ssDNA and DNA ligase such they can be circulated and amplified via rolling circle amplification (RCA). When highly processive DNA polymerases such as Phi29 DNA polymerase is used, the circular DNA template can be replicated hundreds to thousands of times longer and as a result producing ssDNA molecules from hundreds of nanometers to microns in length (see Angewandte Chemie International Edition, 2008, 47, 6330-6337). After the amplification, the replicated DNA can be detected via detection systems. Thus, a visible signal is indicative that the targets of interest are in close proximity. These assays feature the use of several DNA-antibody conjugates as well as enzymes such as DNA ligase and DNA polymerase. These conjugates and enzymes can be expensive, and they also require stringent assay conditions in order for proper function and stability. Furthermore, the approach generates an amplified DNA sequence, while easy to detect, may not remain co-localized with the proximity event detected.
Another assay for investigating protein-protein interactions includes a dual binders (DB) assay, which utilizes a bi-specific detection agent consisting of two Fab fragments with fast off-rate kinetics joined by a flexible linker (Development of Bispecific Molecules for the In Situ Detection of Protein-Protein Interactions and Protein Phosphorylation, Chemistry & Biology 21, 1-12, Mar. 20, 2014). In principle, because the dual binders comprise Fab fragments with fast off-rate kinetics, the dual binders are washed off if only one of the Fab fragments is bound to its epitope (simultaneous cooperative binding of both Fab fragments of the dual binder prevents dissociation of the dual binder and leads to positive staining/visibility). These approaches require the specific development of fab fragments with specific binding kinetics, which makes their implementation to the breadth of targets of interest unreasonable.
In another approach, Grossman et al. describe an assay for the detection of proximal target nucleic acids by the transfer of a reporter group from a donating probe to a nearby accepting probe. The probes are designed to anneal adjacently to complementary segments of the target nucleic acid. The annealing of the probe pair (in close proximity) allows for reactions (e.g., a thio-exchange reaction, etc.) to occur, which facilitates the transfer of the reporter group (e.g., fluorescence quencher) (see Grossman et al., Angew. Chem. Int. Ed. 2008, 47, 7119-7122).
According to another approach, PCT Published Application WO2014/139980, which is incorporated by reference in its entirety for disclosure related to proximity assays and tools for enabling the same, describes the use of a biotin ligase substrate and an enzyme to perform a proximity assay. The method provides detection of target molecules and proximity while maintaining the cellular context of the sample. The use of biotin ligase such as an enzyme from Escherichia coli and peptide substrate such as amino-acid substrate for that enzyme provides for a sensitive and specific detection of protein-protein interactions in FFPE samples. Because biotin ligase can efficiently biotinylate appropriate peptide substrate in the presence of biotin and the reaction can only occur when the enzyme makes physical contact with the peptide substrate, biotin ligase and the substrate can be separately conjugated to two antibodies that recognize targets of interest respectively